Biomedical subjects

S D Perreault

Publications and source records attributed to S D Perreault.

At least 19 recordsLinked to original sources

PubMed1992

Utility of light scatter in the morphological analysis of sperm.

We were able to differentiate the morphologically diverse sperm nuclei of four animal species by using an Ortho flow cytometer to detect the forward light scatter from a red (helium-neon) laser. Cytograms depicting the axial light loss and forward red scatter signals revealed unique, but reproducible, sigmoid distributions that reflected not only interspecies differences in shape and size, but variations in particle refractive index and orientation within the flow cell at the time of analysis. Consequently, we were able to use regional gating of the light scatter cytogram to minimize the influence of orientation on the resolution of the fluorescence signal. We also observed that sperm enlarging as a result of chemically induced decondensation exhibit over time a biphasic shift (increase, then decrease) in light scatter at a species-dependent rate. These results suggest that, without any special adaptations to the flow cytometer, light-scatter parameters can be used to discriminate morphologically different sperm, to enhance the resolution of fluorescence measurements that may otherwise be confounded by variability in radial orientation, and to detect alterations in the rate of a biochemical/biophysical process such as decondensation.

Animals↗

PubMed1992

Use of the fungicide carbendazim as a model compound to determine the impact of acute chemical exposure during oocyte maturation and fertilization on pregnancy outcome in the hamster.

Early pregnancy loss due to acute chemical exposure is difficult to detect and essentially impossible to characterize in humans. Here we use a hamster animal model to identify early pregnancy loss due to an acute chemical exposure to the female during the perifertilization interval. The fungicide carbendazim (methyl 1H-benzimidazole-2-carbamate), a microtubule poison with antimitotic activity, was selected as a model compound because it would be expected to perturb microtubule-dependent events occurring in the oocyte during meiotic maturation and fertilization. Such effects would likely lead to aneuploidy in the zygote with subsequent early pregnancy loss. Female hamsters were given a single oral dose of carbendazim during meiosis I (the afternoon of proestrus) prior to breeding, or during meiosis II (the morning of estrus) following overnight breeding. Pregnancy outcome was assessed on Day 15 (the afternoon before parturition). When given during during meiosis I, carbendazim treatment (750 or 1000 mg/kg body weight) significantly reduced the percentage of pregnant hamsters. In those animals that became pregnant, the average number of live pups was significantly lower at all dosages of carbendazim used (250, 500, 750, and 1000 mg/kg), an effect attributable to both preimplantation and early postimplantation losses. When given early on the morning of estrus, shortly before and during fertilization (0500 or 0600 hr), carbendazim treatment (1000 mg/kg) produced a similar decrease in litter size. This effect disappeared when carbendazim was administered at a slightly later time (0800 or 0900 hr), after the microtubule-dependent events of fertilization have occurred. These results demonstrate that a single exposure to a microtubule poison such as carbendazim at critical times, coincident with microtubule-dependent meiotic events, can result in very early pregnancy loss. Such loss was readily measurable in this animal model and serves as the basis for further mechanistic studies which would be impossible to conduct in humans.

Abortion, Veterinary↗

PubMed1992

Chromatin remodeling in mammalian zygotes.

With sperm-egg fusion at the time of fertilization the gamete nuclei are remodeled from genetically quiescent structures into pronuclei capable of DNA synthesis. Features of this process that are critical to insure the genetic integrity of the zygote and the success of subsequent embryonic development include: oocyte responses that prevent polyspermy; completion of the 2nd meiotic division by the oocyte; exchange of proteins in the sperm nucleus; and, remodelling of the oocyte chromosomes and sperm nucleus into functional pronuclei. Elucidation of the biological and molecular mechanisms underlying zygote formation and chromatin remodeling should enhance our understanding of the potential vulnerability of the zygote to toxicant-induced damage.

Animals↗

PubMed1992

Methods for assessing rat sperm motility.

Computer-assisted sperm analysis (CASA) systems are becoming more widely used. With this spread of technology come more data from toxicology studies, designed to determine if treatment with putative toxicants affects sperm motion parameters. While these CASA methods provide us with more ways to evaluate toxicity and thus perhaps increase our chances of successfully protecting human health, there is also a greater likelihood that different laboratories will use different methods of collecting data on sperm motility. Different systems used with different methods in different laboratories will inevitably generate data that are difficult to compare. In a prospective attempt to address this issue of comparability and limit the problems, a group of individuals using CASA systems to analyze rat sperm motility convened to discuss methodologic issues, share data, and try to reach a consensus about methods for performing these studies. This article shares those meetings and data in the hope that common methods will enhance interlaboratory comparisons.

Animals↗

PubMed1991

Validation of protocols for assessing early pregnancy failure in the rat: clomiphene citrate.

A battery of protocols for the assessment of maternally mediated toxicity during early pregnancy in the rat is being evaluated for its utility in detecting and defining mechanisms of early pregnancy failure. In this report, clomiphene citrate (CC), an estrogen agonist/antagonist, was used as a model compound in this evaluation process. The protocols involve dosing rats with CC during, and evaluation of multiple endpoints following, (a) the first 8 days of pregnancy; (b) early pseudopregnancy, accompanied by decidual induction; and (c) the pre- and postimplantation intervals of early pregnancy. In addition, the effect of CC on embryo transport rate was assessed. Eight days of dosing with CC during early pregnancy produced a dose-dependent reduction in the number of implantation sites seen on Day 9, concomitant with alterations in maternal hormonal parameters. No effect on the decidual cell response was found, indicating that the mechanism of fertility reduction was not mediated via compromised uterine decidualization. The preimplantation period was highly vulnerable to the toxic effects of CC while no postimplantation resorptions were seen. When embryo transport rate was measured, a statistically significant embryo transport rate acceleration was detected, but this was insufficient to account for all of the observed preimplantation loss. The data suggest an effect of CC on embryo viability, on the embryo's ability to implant, or on the reproductive tract resulting in compromised embryo survival as the potential mechanism(s) mediating CC-induced early pregnancy failure. The battery of protocols was thus successful in identifying CC as a reproductive toxicant and in elucidating the causes of the observed early pregnancy failure.

Animals↗

PubMed1991

Use of bromoergocryptine in the validation of protocols for the assessment of mechanisms of early pregnancy loss in the rat.

Validated protocols for evaluating maternally mediated mechanisms of early pregnancy failure in rodents are needed for use in the risk assessment process. To supplement previous efforts in the validation of a panel of protocols assembled for this purpose, bromoergocryptine (BEC) was used as a model compound because it is known to inhibit pituitary prolactin secretion. BEC was tested using the early pregnancy protocol (EPP), the decidual cell response technique (DCR), the pre- vs postimplantation protocol (PPP), and embryo transport rate analysis (ETRA). These protocols evaluate the effects of chemicals on multiple endpoints following exposure during (a) the first 8 days of pregnancy, (b) early pseudopregnancy accompanied by decidual induction, (c) the pre- and postimplantation intervals of early pregnancy, or (d) the period of embryo transport. In the EPP, dosing with BEC during Days 1-8 of pregnancy reduced the number of implantation sites found on Day 9 as well as serum progesterone. The DCR technique revealed a dose-dependent inhibition of decidual growth concomitant with decreased serum progesterone as a result of BEC treatment. A modified DCR technique using hormone-supplemented ovariectomized rats demonstrated that BEC did not impair decidual growth in the presence of adequate progestogenic support. Pre- vs postimplantation dosing indicated that implantation is vulnerable to BEC effects at least through Day 4. BEC had no effect on embryo transport rate. Data from these protocols identified BEC as having adverse maternal effects during early pregnancy. While the pituitary was not identified by these protocols as the site of BEC's primary action, the protocols did identify a reduction in serum progesterone and impaired uterine function as toxicological mechanisms mediating the reduced fertility seen following BEC exposure.

Abortion, Veterinary↗

PubMed1991

Rat sperm motility analysis: methodologic considerations.

The objective of these studies was to optimize conditions for computer-assisted sperm analysis (CASA) of rat epididymal spermatozoa. Methodologic issues addressed include sample collection technique, sampling region within the epididymis, type of diluent medium used, and sample chamber depth. In addition, sources of variation were identified and accuracy of the analysis was examined. All samples in this report were analyzed using a Hamilton Thorn Motility Analyzer (HTM-2000; Hamilton Thorn Research, Danvers, MA). We found that allowing the sperm to swim out from cuts made in the distal cauda epididymidis yielded samples with percentages of motile sperm 60% higher than samples collected using an aspiration method. Furthermore, sperm isolated from the distal cauda epididymidis exhibited slightly but significantly greater percentages of motile sperm and swimming speeds than sperm isolated from the proximal cauda epididymidis. Of the four motility media examined, all maintained a high percentage of motile sperm over an hour-long incubation period, but Medium 199 and modified Hanks' Balanced Salt supported substantially greater sperm velocity than Dulbecco's Phosphate Buffered Saline (with Ca++ and Mg++), with or without glucose. Motility and velocity endpoints were comparable in 200-, 100-, or 40-micron deep chambers, but significantly lower in 20-micron-deep chambers. Since these and presumably other variables in the preparation and analysis of rat sperm do influence the assessed motility endpoints, it is important to standardize these methods and to consider these issues when interpreting CASA data.

Animals↗

PubMed1991

Thermostability of sperm nuclei assessed by microinjection into hamster oocytes.

Nuclei isolated from spermatozoa of various species (golden hamster, mouse, human, rooster, and the fish tilapia) were heated at 60 degrees-125 degrees C for 20-120 min and then microinjected into hamster oocytes to determine whether they could decondense and develop into pronuclei. Mature, mammalian sperm nuclei, which are stabilized by protamine disulfide bonds, were moderately heat resistant. For example, they remained capable of pronucleus formation even after pretreatment for 30 min at 90 degrees C. Indeed, a temperature of 125 degrees C (steam) was required to inactivate hamster sperm nuclei completely. On the other hand, nuclei of rooster and tilapia spermatozoa and those of immature hamster and mouse spermatozoa, which are not stabilized by protamine disulfide bonds, were sensitive to heating; although some of them decondensed after exposure to 90 degrees C, none formed male pronuclei. Furthermore, nuclei of mature hamster sperm became heat labile when they were pretreated with dithiothreitol to reduce their protamine disulfide bonds. These observations suggest that the thermostability shown by the nuclei of mature spermatozoa of eutherian mammals is related to disulfide cross-linking of sperm protamines.

Animals↗

PubMed1990

Methoxychlor accelerates embryo transport through the rat reproductive tract.

The estrogenic pesticide methoxychlor (MXC) is known to reduce implantation, and, in our previous work, this reduction has been attributed to a direct effect on uterine function. The present study was designed to investigate the effect of MXC on embryo transport rate, another phenomenon that is vulnerable to estrogenic effects. MXC was administered by gavage, at 0, 100, 200, and 500 mg/kg/day, to groups of rats on Days 1-3 of pregnancy (Day 0 = sperm positive), and the distribution of embryos in the oviducts and uteri of animals was assessed at five time intervals prior to implantation. No effect of MXC was detected by the afternoon of Day 1. On Days 2 and 3 of pregnancy, 200 and 500 mg/kg/day MXC were found to accelerate embryo transport into the uterus; the 500 mg/kg/day dosage also reduced the total number of embryos recovered from the tract. On the third day, 100 mg/kg/day MXC also accelerated embryo transport to the uterus and 200 mg/kg/day MXC reduced total embryo recovery. Until the afternoon of Day 3, most control embryos remained in the oviduct. These data demonstrate that MXC produces a dose-dependent acceleration of embryo transport through the female reproductive tract. When compared with previous work, the current data indicate that such an acceleration is the primary cause of MXC-induced preimplantation embryonic loss when exposure occurs after fertilization.

Animals↗

PubMed1990

Acute inhalation exposure to epichlorohydrin transiently decreases rat sperm velocity.

The effect of inhaled epichlorohydrin on rat sperm motility characteristics was evaluated. Male F-344 rats were exposed to 100 ppm epichlorhydrin via inhalation for 4 hr in the morning of Day 0 and killed immediately and on Days 1, 2, 6, and 14 postexposure. Videotapes of cauda epididymal sperm were analyzed (300-350 sperm/sample) with a Hamilton Thorn motility analyzer (HTM-2000, Hamilton Thorn Research, Danvers, MA). Epichlorohydrin did not affect the percentage of motile sperm at any time. However, transient changes in sperm velocity were found. On Day 1 postexposure mean progressive (straight line) and mean path (smoothed curvilinear) velocity were significantly decreased to 80 and 85% of control, respectively. The progressive velocities of sperm from both control and treated rats were normally distributed, indicating a general effect of epichlorohydrin on all sperm as opposed to a more severe effect on a specific sperm subpopulation. Sperm velocity was not significantly affected at later times. Other endpoints (testis and epididymis weights, testicular spermatid counts, and cauda epididymal sperm reserves) were unaltered by epichlorohydrin. Thus, inhaled epichlorohydrin at 100 ppm produced specific, transient decreases in rat sperm velocity. Furthermore, computer-assisted sperm analysis was able to detect these relatively subtle, toxicant-induced changes in rat sperm velocity, demonstrating the utility of this technology in reproductive toxicology studies.

Administration, Inhalation↗

PubMed1990

Reproductive toxicity of a single dose of 1,3-dinitrobenzene in two ages of young adult male rats.

These studies evaluated the reproductive response and the possible influence of testicular maturation on the reproductive parameters, in male rats treated with 1,3-dinitrobenzene (m-DNB). Young adult male rats (75 or 105 days of age) were given a single oral dose of 0, 8, 16, 24, 32, or 48 mg/kg of m-DNB and killed at 14 days post-treatment. Mortality and neurotoxicity were observed at 48 mg/kg, but only in the older animals. Epididymis weight, testicular sperm head counts, cauda sperm reserves, and sperm morphology were affected at 16 and 24 mg/kg and higher in the older and younger animals, respectively. Testis weight and sperm motility were affected at 24 mg/kg and higher in both age groups. Histologic changes included maturation depletion of mid and late spermatids at 16 mg/kg and higher, atrophy of a few to many seminiferous tubules at 24 mg/kg and higher, and immature germ cells in the epididymis. The movement and/or mixing of luminal elements in the epididymis appeared to be influenced by severe testicular effects. In separate groups given only the 48 mg/kg dosage, fertilizing ability was lost by 5-6 weeks post-treatment and several animals failed to recover in 5 months. In the breeder males, minimal to extensive degrees of seminiferous tubule atrophy and sloughed germ cells in the epididymis were still present after 175 days. The studies indicated that the lowest dosage to produce reproductive changes was 16 mg/kg with a no-effect level of 8 mg/kg. A few animals suffered protracted or permanent reproductive damage. Since the older animals were more susceptible to both the general and the reproductive toxicity of m-DNB, the less severe reproductive changes in the younger animals cannot be attributed solely to maturational differences in the testis.

Aging↗

PubMed1990

Importance of glycolysable substrates for in vitro capacitation of human spermatozoa.

To investigate the importance of glycolysable substrate for supporting the ability of human sperm to capacitate and penetrate oocytes in vitro, washed spermatozoa were incubated with or without various sugars in BWW culture medium containing pyruvate and lactate. Sperm penetration was assayed using zona-free hamster oocytes. After an 18-h preincubation, glucose (1 mg/ml) supported higher penetration of sperm into oocytes than either mannose or fructose (60.7% vs. 28.2% or 21.5%, respectively) at the same concentration. Penetration was even lower when medium contained the nonmetabolizable sugar galactose (2.1% at 1 mg/ml). On the other hand, higher concentrations (5 or 10 mg/ml) of glucose, but not fructose, suppressed penetration, provided the glucose was present throughout the 18-h preincubation. When caffeine, a stimulant of glycolysis in human sperm, was present along with glucose, sperm penetration was enhanced, but only after 6 h of sperm preincubation. This effect was not observed in glucose-free medium, however, where penetration remained low over a 10-h incubation period. In these experiments, the percentage of motile sperm was unaffected by treatment, but the quality of motility was diminished in the absence of glucose. We conclude that stimulation of glycolysis may promote capacitation of human spermatozoa in vitro and that optimization of penetrating ability of sperm is dependent upon both the type and concentration of glycolysable sugar present.

Animals↗

PubMed1989

Unilateral depletion of testicular glutathione levels in the rat following intratesticular injections of diethylmaleate and buthionine sulfoximine.

A method was developed to selectively deplete glutathione (GSH) in a single rat testis. Using intratesticular injections of a mixture of two GSH-depleting agents, diethylmaleate and buthionine sulfoximine, testicular GSH levels were decreased to 33-54% of control 2 hr after injection and remained suppressed for 24 hr. GSH levels in the contralateral testis and liver were not affected by this treatment. Comparisons between GSH-depleted and vehicle-injected (contralateral) testes, evaluated 2 weeks later, showed that although testis and epididymal weights and cauda epididymal sperm reserves were slightly reduced (to greater than or equal to 90% of controls), no changes were seen in testicular spermatid counts or in the morphology or motility of cauda epididymal sperm. An increase in histologically abnormal tubules localized to the injection site occurred in some GSH-depleted testes; however, the proportion of normal tubules containing step 19 spermatids was not affected. Thus, intratesticular injections of GSH-depleting agents selectively lowered GSH levels in the treated testis, with minimal adverse effects. This protocol can now be applied to investigate specific roles of GSH in the testes, particularly with regard to the possible modulation of the effects of testicular toxicants.

Animals↗

PubMed1989

The value of multiple endpoint data in male reproductive toxicology: revelations in the rat.

To explore the relationship between sperm measures and reproductive success in the laboratory rat, a review has been conducted of results of several male reproductive toxicology studies containing both fertility and multiple endpoint data. Comparisons are made between subchronic and acute exposure studies, emphasizing for each approach the value of multiple endpoint data. Further, the choice of endpoints in male reproductive toxicology is discussed with select examples of endpoints for which an understanding is evolving. We conclude that sperm measures are far more sensitive indicators of reproductive organ impairment than is the measurement of fertility, and that evaluation of multiple endpoints in acute studies is a key step in determining mechanisms of toxicant action that are critical for accurate interspecies risk extrapolation.

2,4-Dinitrophenol↗

PubMed1988

Importance of glutathione in the acquisition and maintenance of sperm nuclear decondensing activity in maturing hamster oocytes.

Sperm nuclear decondensing activity in mammalian oocytes is dependent upon the maturational state of the oocyte. It is maximal in mature, metaphase II oocytes and minimal or absent in immature germinal vesicle (GV) and fertilized pronuclear oocytes. Previous studies suggested that this difference may be due to the relative ability of an oocyte to reduce the protamine disulfide bonds in the sperm nucleus. The results of this study show that mature hamster oocytes contain significantly more glutathione (GSH), about 8 mM, and hence more disulfide reducing power, as compared with GV (4 mM) or pronuclear (6 mM) oocytes. Furthermore, the acquisition of sperm nuclear decondensing activity by maturing oocytes can be prevented or delayed by blocking GSH synthesis with L-buthionine-S,R-sulfoximine during the early stages of oocyte maturation. This is the first evidence that modulation of GSH levels during oocyte maturation and fertilization may be a mechanism by which sperm nuclear decondensing activity is regulated.

Animals↗

PubMed1988

Interspecies differences in the stability of mammalian sperm nuclei assessed in vivo by sperm microinjection and in vitro by flow cytometry.

To assess the structural stability of mammalian sperm nuclei and make interspecies comparisons, we microinjected sperm nuclei from six different species into hamster oocytes and monitored the occurrence of sperm nuclear decondensation and male pronucleus formation. The time course of sperm decondensation varied considerably by species: human and mouse sperm nuclei decondensed within 15 to 30 min of injection, and chinchilla and hamster sperm nuclei did so within 45 to 60 min, but bull and rat sperm nuclei remained intact over this same period of time. Male pronuclei formed in oocytes injected with human, mouse, chinchilla, and hamster sperm nuclei, but rarely in oocytes injected with bull or rat sperm nuclei. However, when bull sperm nuclei were pretreated with dithiothreitol (DTT) in vitro to reduce protamine disulfide bonds prior to microinjection, they subsequently decondensed and formed pronuclei in the hamster ooplasm. Condensed rat spermatid nuclei, which lack disulfide bonds, behaved similarly. The same six species of sperm nuclei were induced to undergo decondensation in vitro by treatment with DTT and detergent, and the resulting changes in nuclear size were monitored by phase-contrast microscopy and flow cytometry. As occurred in the oocyte, human sperm nuclei decondensed the fastest in vitro, followed shortly by chinchilla, mouse, and hamster and, after a lag, by rat and bull sperm nuclei. Thus species differences in sperm nuclear stability exist and appear to be related to the extent and/or efficiency of disulfide bonding in the sperm nuclei, a feature that may, in turn, be determined by the type(s) of sperm nuclear protamine(s) present.

Animals↗

PubMed1987

DNA synthesis following microinjection of heterologous sperm and somatic cell nuclei into hamster oocytes.

We have investigated the ability of the hamster oocyte to initiate DNA synthesis in nuclei differing in basic protein content. DNA synthesis was studied by autoradiography in oocytes that had been incubated in 3H-thymidine after being parthenogenetically activated by sham microinjection, or microinjected with hamster, mouse, rabbit, or fish sperm nuclei, or hamster hepatocyte nuclei. Within 6 hr of sham or nucleus microinjection, nuclei of each type underwent transformation into pronuclei and synthesized DNA. These results demonstrated that the hamster egg can access and utilize its own and each type of template provided, whether homologous or heterologous. However, pronuclei derived from hamster sperm nuclei were more likely to be synthesizing DNA at 6 hr than pronuclei derived from sperm nuclei of other species. We conclude that the mechanisms employed by the hamster oocyte to transform hamster sperm nuclei into pronuclei and to effect DNA synthesis in these nuclei are not specific for the hamster sperm nucleus. Nevertheless, these mechanisms apparently operate more efficiently when the hamster sperm nucleus, rather than a heterologous sperm nucleus, is present.

Animals↗

PubMed1987

Incidence of chromosome aberrations in mammalian sperm stained with Hoechst 33342 and UV-laser irradiated during flow sorting.

The separation of two sperm populations is possible using the technique of flow sorting, provided that a significant difference exists in the DNA content of X- and Y-bearing sperm. In order to ascertain whether or not chromosome damage was induced in sorted sperm, chromosome preparations were made from isolated sperm that had been microinjected into hamster eggs. While egg chromosomes exhibited a low frequency of chromosome aberrations, ranging from 4 to 7%, a large proportion of sperm cells exhibited chromosome damage. Between 29% of unstained and unsorted sperm and 38% of stained and unsorted sperm exhibited some type of chromosomal abnormality and this proportion increased to 50% in sorted sperm. If only damaged sperm nuclei are considered, the two unsorted sperm groups had a mean of 0.6 breaks, 0.8 triradial exchanges, and 0.2 quadriradial exchanges per nucleus. However, sorted sperm, which were stained with a fluorochrome and exposed to UV-laser irradiation, exhibited a mean of 2.9 breaks, 2.6 triradial, and 1.9 quadriradial exchanges per nucleus in which damage occurred. These observations indicate that the treatments and manipulations to which sperm nuclei are subjected during flow sorting cause chromosomal aberrations, and that exposure of the cells to UV-laser irradiation contributes substantially to the chromosome damage observed.

Animals↗